Most plants produce and store starch. These plants have a starch synthesis pathway for starch production. The amount of starch produced varies with the type of plant. The most commonly known starch producing plants are the cereal grains. These cereals include rice, maize, sorghum, barley, wheat, rye, and oats. Additionally, the potato family, including the sweet potatoes and certain fruits, like the banana, are known as starch producing.
Starch is an important end-product of carbon fixation during photosynthesis in leaves and is an important storage product in seeds and fruits. In economic terms, the starch produced by the edible portions of three grain crops, wheat, rice and maize, provide approximately two-thirds of the world's food calculated as calories.
Starch from plants is used in various ways. For example, it can be extracted and used for cooking and food processing. Starch can be left in the grain or plant and used for animal and human consumption. Starch can also be used in the distillation process for processing alcohols, for example, starch can be converted into ethanol. Additional starch can convert to high-fructose syrup and other industrial components.
Starch is defined in the dictionary as a granular solid which is chemically a complex carbohydrate which is used in adhesives, sizes, foods, cosmetics, medicine, etc. More generally, starch is comprised of amylose and amylopectin. Amylose and amylopectin is synthesized in the plastid compartment (the chloroplast in photosynthetic cells or the amyloplast in non-photosynthetic cells). Different plants generate differing proportions of amylopectin and amylose. Furthermore, the different branching patterns of amylopectin and different chain lengths of amylose and amylopectin chains gives rise to different starch properties. Thus, the fine structure of amylose and amylopectin is different in different plants so that the branching patterns and chain-lengths vary considerably resulting in new and novel properties which are useful in different applications. Until now there have been four ways of making starches with special properties: (i) using starches extracted from different plant species, (ii) using starches extracted from mutant lines of particular plants, (iii) using natural and mutant starches which had been chemically modified, and (iv) using natural and mutant starches which had been physically modified. In all cases the new starches were valuable because of the special properties provided for by the new starch type.
It is known that mutant genes in plants affect the properties of the starch. A variety of starch related mutant genes in maize have been identified and some have been cloned. These mutant genes were named according to the physical appearance (phenotype) of the maize kernel or the properties of the starch. These recessive mutant genes include waxy (wx), sugary (su) [which includes but is not limited to sugary-1 (su1), sugary-2 (su2), sugary-3 (su3), sugary-4 (su4)] dull (du), amylose extender (ae), horny (h), shrunken (sh) which includes, but is not limited to, shrunken-1 (sh-1), shrunken-2 (sh-2). Some of these recessive gene mutants produce an isoform of a known enzyme in the starch synthesis pathway. The recessive mutant alleles of these genes result in a complete or nearly complete reduction in the activity of a specific isoform of one enzyme (hereinafter defined as complete reduction of enzyme isoform activity) in the pathway when homozygous in a plant or when expressed in sufficient levels in a transgenic plant. This change in the starch synthesis pathway causes the formation of starches with different properties.
Several crop varieties are known which produce different types of starch. The type of quality of starch makes it suitable for certain purposes, including particular methods of processing or particular end-uses. Naturally-occurring maize mutants produce starches of differing fine structure suitable for use in various food products and other applications. Although known mutants produce altered starch, some of these lines are not suitable for crop breeding and/or for the farmers' purposes. For example, they can give relatively poor yields, and/or are difficult to process and/or can have poor germination.
In order to generate different starches, single and double mutant plants have been bred. A single mutant is a plant that is homozygous for one recessive mutant gene. For example, waxy maize, waxy rice, waxy barley, and waxy sorghum have the homozygous mutant waxy (wx) gene. Whilst starches from waxy genotypes have very little or no amylose, another mutation known as amylose extender (ae) results in starch with high amylose. A double mutant is a single plant that has homozygous (or full expression) of two recessive mutant genes. For example, the wxfl1 double mutant is taught in U.S. Pat. No. 4,789,738. Many other novel starches have been provided in other starch patents in which double or triple mutants are generated (for example, U.S. Pat. Nos. 4,789,557, 4,790,997, 4,774,328, 4,770,710, 4,798,735, 4,767,849, 4,801,470, 4,789,738, 4,792,458 and 5,009,911 which describe naturally-occurring maize mutants producing starches of differing fine structure suitable for use in various food products). The present invention is highly surprising in light of these applications because it produces altered starch and does not require double or triple mutants.
Normal starch is defined as starch which is not chemically modified (by people) or which is produced from a plant that has the expected genes (wild type) regulating the starch synthesis pathway. For ease of reading, double lower-case letters, for example aa, shall refer to a homozygous recessive mutant gene, double upper-case letters, for example AA, shall refer to a homozygous non-mutant gene (wild type), and one upper-case and one lowercase letter, for example Aa, shall refer to a non-homozygous set of genes, one mutant, one non-mutant. Different letters in the same size shall mean different genes; "aa/bb" would be a double mutant; "aa/bB" would be a single homozygous mutant gene and a heterozygous mutant gene in the genome of the plant. For purposes of this application, the order of any three letters on one side of the slash can be interchanged and will not define the parent that donated the gene. For example, AAa/bbB is defined to be equivalent to aAA/bBb, AaA/Bbb, AaA/bBb, aAA/Bbb, and the like.
Although maize plants and the embryo are diploid, maize endosperm is triploid. The endosperm genotype has two gene doses which are inherited from the female plant portion and one gene dose which is inherited from the pollen or male plant portion. Thus, if a single mutant plant "aa" is used as the female and crossed to a non-mutant plant "AA" male, then the endosperm in the kernel of this female plant would be "aaA". If a non-mutant plant "AA" is crossed to a mutant plant "aa" with the non-mutant as the female, the endosperm on the kernel of the female plant will be "Aaa", because two gene doses come from the female and one from the male plant. Classic teaching is that the mutant gene is recessive and the non-mutant is dominant; therefore, the starch produced by a plant having the following gene doses in the endosperm "aaA" or "AAA" or "AAa" results in the normal starch in the expected amounts. However, the endosperm of a homozygous mutant plant "aa" acting as the female crossed to a homozygous mutant plant "aa" acting as the male plant results in the endosperm having the gene dosage "aaa". This endosperm gives starch with different properties from normal starch. Likewise, the starch from a double mutant having of an endosperm which is "aaa/bbb" shows differences in starch properties from normal starch. These starch differences are useful in that they can replace chemically modified starches or be used with or in foodstuffs or as grain in alcohol production or in general starch industrial applications.
Clearly, it appears that production of grain having starch with different physical properties of starch requires the crossing of two mutated plants to generate grain which is homozygous recessive for both genes. Mutant plants are less predictable than standard plants.
There is a recurrent problem with the production of grain and extraction of starch from double mutant hybrids and/or inbreds and some single mutants. The amount of starch produced is usually less than the amount of starch produced by the non-mutant plant, there is also a loss in starch granule size and/or starch granule integrity. This problem with known double mutant lines which produce structurally-altered starch in which the quantity of starch produced in the crop is relatively low can result in poor germinability of the seed. Furthermore, the reduced starch yield of the seed appears to be unavoidable since the mutations cause the normal starch synthesis functioning of the cells to be disrupted. There remains a need for a way to produce grain having structurally altered starch structurally altered starch or altered properties without a significant loss of yield or reduced starch granule size or integrity.